The past decade has been characterized by significant advances in the science of cloning, and has witnessed the birth of a cloned sheep, i.e. “Dolly” (Roslin Bio-Med), a trio of cloned goats named “Mira” (Genzyme Transgenics) and over a dozen cloned cattle (Advanced Cell Technology or ACT). Most recent additions to the clone family include pigs (PPL Therapeutics) and mice (University of Hawaii Medical School). Scientists at ACT have also demonstrated successful cross-species nuclear reprogramming by the birth of a cloned guar produced using a bovine recipient oocyte. For example, see U.S. patent application Ser. No. 09/685,062, incorporated by reference herein in its entirety. Furthermore, cloning technology has also advanced such that a mammal may now be cloned using the nucleus from an adult, differentiated cell, which scientists now know undergoes “reprogramming” when it is introduced into an enucleated oocyte. See U.S. Pat. No. 5,945,577, incorporated herein by reference in its entirety.
The showing that an embryo and embryonic stem cells may be generated using the nucleus from an adult differentiated cell has exciting implications for the fields of organ, cell and tissue transplantation. There are currently thousands of patients waiting for a suitable organ donor, who face the problems of both availability and incompatibility in their wait for a transplant. By using a differentiated cell from a patient in need of a transplant to generate embryonic stem cells, and inducing these to differentiate into characterized populations of the cell type required in the transplant, the problem of transplantation rejection and the dangers of immunosuppressive drugs could be precluded. This prospect is now known to many as “therapeutic cloning,” or “adult cell reprogramming” so as to distinguish it from “reproductive cloning” and provides a moral boundary as the reach of cloning extends toward the realm of human beings. Lanza et al., September 1999, Human therapeutic cloning, Nat. Med. 5(9): 975-7.
Conscious of the promise of therapeutic cloning, scientists are seeking to understand how to efficiently direct the differentiation of totipotent and pluripotent stem cells into particular cell types and tissues, while at the same time deterring their differentiation into unwanted cells and tissues. Controlled, specific direction of cell differentiation will come from deciphering the factors and signals that control embryonic development. The alternative, e.g., the random differentiation of embryonic cells and subsequent dissection of desired tissues, is both impractical and morally unacceptable for human therapy.
As used herein, a “stem cell” is a cell that has the ability to divide for indefinite periods in culture and to give rise to daughter cells of one or more specialized cell types.
As used herein, an “embryonic stem cell” (ES-cell) is a cell line with the characteristics of the murine embryonic stem cells isolated from morulae or blastocyst inner cell masses (as reported by Martin, G., Proc. Natl. Acad. Sci. USA (1981) 78:7634-7638; and Evans, M. and Kaufman, M., Nature (1981) 292: 154-156) i.e., ES cells are immortal and capable of differentiating into all of the specialized cell types of an organism, including the three embryonic germ layers, all somatic cell lineages, and the germ line.
As used herein, an “embryonic stem-like cell” (ES-like cell) is a cell of a cell line isolated from an animal inner cell mass or epiblast that has a flattened morphology, prominent nucleoli, is immortal, and is capable of differentiating into all somatic cell lineages, but when transferred into another blastocyst typically does not contribute to the germ line. An example in the primate “ES cell” reported by Thomson et al. (Proc. Natl. Acad. Sci. USA. (1995) 92:7844-7848)
As used herein, “inner cell mass-derived cells” (ICM-derived cells) are cells derived from isolated ICMs or morulae before they are passaged to establish a continuous ES or ES-like cell line.
As used herein, an “embryonic germ cells” (EG cells) is a cell of a line of cells obtained by culturing primordial germ cells in conditions that cause them to proliferate and attain a state of differentiation similar, though not identical to embryonic stem cells. Examples are the murine EG cells reported by Matsui, et al, 1992, Cell 70: 841-847 and Resnick et al, Nature. 359: 550-551. EG cells can differentiate into embryoid bodies in vitro and form teratocarcinomas in vivo (Labosky et al., Development (1994) 120:3197-3204). Immunohistochemical analysis demonstrates that embryoids produced by EG cells contain differentiated cells that are derivatives of all three embryonic germ layers (Shamblott et al., Proc. Nat. Acad. Sci. U.S.A. (1998) 95:13726-13731).
As used herein, a “totipotent” cell is a stem cell with the “total power” to differentiate into any cell type in the body, including the germ line following exposure to stimuli like that normally occurring in development. An example of such a cell is an ES cell, an EG cell, an ICM-derived cell, or a cultured cell from the epiblast of a late-stage blastocyst.
As used herein, a “nearly totipotent cell” is a stem cell with the power to differentiate into most or nearly all cell types in the body following exposure to stimuli like that normally occurring in development. An example of such a cell is an ES-like cell.
As used herein, a “pluripotent cell” is a stem cell that is capable of differentiating into multiple somatic cell types, but not into most or all cell types. This would include by way of example, but not limited to, mesenchymal stem cells that can differentiate into bone, cartilage and muscle; hemotopoietic stem cells that can differentiate into blood, endothelium, and myocardium; neuronal stem cells that can differentiate into neurons and glia; and so on.
As used herein, “differentiation” refers to a progressive, transforming process whereby a cell acquires the biochemical and morphological properties necessary to perform its specialized functions.
As used herein, a “marker” is a characteristic or feature of a cell that is indicative of a particular cellular state. Typically, a marker is a biochemical entity that changes state in a detectable manner when the cell enters or leaves a particular state. For example, a marker may be a DNA sequence encoding a product that is detectable (e.g., a specific mRNA, or a fluorescent or antigenic protein) or has detectable activity (e.g., a protein conferring antibiotic resistance or a chromogenic enzyme such as lacZ). When copies of the marker DNA sequence are randomly inserted into the genomic DNA of a cell, some copies may be inserted proximal to a promoter in the correct orientation and in-frame such that activation of the promoter results in transcription of the marker DNA sequence and synthesis of the detectable product that it encodes. Detection of the marker then identifies the cell as one that contains the marker gene in a transcriptionally active genetic locus. The term “marker” as used herein may refer to a marker gene, or to a marker RNA or protein encoded by such a gene.
Directed Differentiation of Stem Cells
Totipotent and nearly totipotent embryo-derived stem cells can be induced to differentiate into a wide variety of cell types, some of which are needed for cell therapy. For example, Anderson et al. demonstrated that inner cell masses (ICM) and embryonic discs from bovine and porcine blastocysts will develop into teratomas containing differentiated cell types from ectodermal, mesodermal and endodermal origins when transplanted under the kidney capsule of athymic mice. Animal Repro. Sci. 45: 231-240 (1996). Thomson et al. reported that primate ES cells are capable of differentiating into trophoblast and derivatives of the three embryonic germ layers, and describe transplanting primate ES cells into muscles of immunodeficient mice to generate teratomas that also contain cells of the three embryonic germ layers, including tissues resembling neural tube, embryonic ganglia, neurons, and astrocytes (APMIS (1998) 106(1):149-156). ES cells of mice (Lee et al., Nature Biotech. (2000) 18:675-679), cynomolgus monkeys (Macaca fascicularis) (Cibelli et al., Science (2002) 295:819), and humans (Zhang et al., Nature Biotech. (2001) 19:1129-1133) can be cultured in vitro to generate embryoids that contain cells of all three germ layers, including neural precursor cells that test positive for nestin (an intermediate filament protein produced in the developing central nervous system and widely used as a marker for proliferating neural progenitor cells in the nervous system). Pluripotent stem cells can be isolated from ES and EG cell-derived teratomas and embryoids and exposed to conditions that induce them to differentiate into specific cell types that are useful for cell therapy. For example, nestin-positive neural stem cells isolated from human embryoids can be cultured under conditions that induce their differentiation into the three major cell types of the central nervous system (see Zhang et al. (2001) p. 1130).
The foregoing reports describe the derivation of precursor or differentiated cells that appear to arise randomly or spontaneously in embryoids and teratomas generated from totipotent ES and EG cells. Production of a characterized population of differentiated cells by these methods therefore requires isolating the differentiated cells of interest, or their precursors, from other types of cells in an embryoid or teratoma. Presently, there is strong interest in identifying chemical, biological, and physical agents or conditions that induce totipotent or nearly totipotent cells such as ES and EG cells to differentiate directly into the desired differentiated cells, in order to develop efficient methods for producing characterized populations of differentiated cells that are useful for cell therapy.
In U.S. Pat. No. 5,733,727, Field described plating murine ES cells onto uncoated petri dishes and culturing them in medium that is free of leukemia inhibitory factor (LIF), an inhibitor of differentiation, to generate patches of cardiomyocytes that exhibit spontaneous contractile activity (col. 12, lines 63-67). Field also described a useful method for purifying cells induced to differentiate into a specific cell type from other types of cells present in the culture: the parental ES cells are cotransfected with a pGK-HYG (hygromycin) plasmid and a plasmid containing a MHC-neor fusion gene—an α-cardiac myosin heavy chain (MHC) promoter operably linked to a neor gene that confers resistance to neomycin. The pGK-HYG plasmid provides selection for transfected cells, while the MHC-neor gene permits a second round of selection of the differentiated cells—incubation in the presence of G418 eliminates non-cardiomyocyte cells in which the MHC promoter is inactive (see col. 12, lines 63-67). The disclosure of U.S. Pat. No. 5,733,727 is incorporated herein by reference in its entirety.
Schuldiner et al. described a systematic approach to analyzing the differentiation of ES-derived cells in response to different growth factors. They cultured human ES cells to generate embryoids, dissociated the embryoids and cultured the cells as a monolayer in the presence of one of eight different growth factors. The differentiation induced by the growth factors was examined by monitoring changes in the cells' morphologies, and by RT-PCR (reverse transcription—polymerase chain reaction) analysis of the expression of a panel of 24 cell-specific genes in the parental ES cells, embryoid cells, and the dissociated embryoid cells cultured in the presence or the absence of one of the eight growth factors. Schuldiner et al. reported that each of the growth factors appeared to induce expression of different subset of the 24 marker genes that were analyzed; and that the growth factor-treated cultures were relatively homogenous, often containing only one or two cell types, whereas the dissociated embryoid cells cultured in the absence of a growth factor spontaneously differentiated into many different types of colonies. The growth factors appeared to act more by inhibiting than by inducing the differentiation of specific cell types, and none of the growth factors tested directed a completely uniform and singular differentiation of cells, and suggesting that direction of formation of specific cell types will require combinations of factors including those that inhibit undesired pathways and those that induce differentiation of specific cell types. (See Proc. Natl. Acad. Sci. USA (2000) 97(21): 11307-12). Paquin et al. described culturing murine P19 ES cells under conditions resulting in formation of aggregates of cells, some of which differentiated into beating cardiomyocytes (Proc. Nat. Acad. Sci. (2002) 99(14):9550-9555). Reubinoff et al. described manipulating the conditions in which human ES cells were cultured to induce their differentiation directly into neural precursors that could then be induced to differentiate into derivatives of the three neural lineages, neuronal cells, glial cells, and astrocytes (Nature Biotechnology (2001) 19:1134-1139). Kelly et al. have shown that changes in gene expression in ES cells in response to retinoic acid are highly reproducible (Mol. Reprod. Dev. (2000) 56(2): 113-23), a result that implies that growth factor-directed differentiation of embryonic cells is dependably reproducible.
Other groups have had success in using a negative approach to identify factors necessary for the differentiation of ES cells into certain cell types. For instance, Henkel and colleagues reported that the transcription factor PU.1 is essential for macrophage development from embryonic stem cells by showing that ES cells containing a homozygous knockout of the PU.1 gene failed to differentiate into macrophages (see Henkel et al., Blood (1996) 88(8): 2917-26). Similarly, Dunn and colleagues demonstrated that knockout embryoid bodies containing a targeted disruption of the phosphatidylinositol glycan class A (Pig-a) gene failed to develop secondary hematopoietic colonies and demonstrated a grossly aberrant morphology (see Dunn et al., Proc. Natl. Acad. Sci. USA (1996) 93(15): 7938-43).
Directed differentiation has also been demonstrated successfully in pluripotent adult stem cells. For instance, U.S. Pat. No. 5,942,225 to Bruder et al. describes the lineage-directed induction of human mesenchymal stem cell differentiation by exposing such stem cells to a bioactive factor or combination of factors effective to induce differentiation either ex vivo or in vivo. Mesenchymal stem cells are more differentiated than embryonic stem cells and only differentiate into lineages including osteogenic, chondrogenic, tendonogenic, ligamentogenic, myogenic, marrow stromagenic, adipogenic and dermogenic lineages. Similarly, U.S. Pat. No. 5,851,832 to Weiss et al. describes the in vitro proliferation and differentiation of neural stem cells following exposure of the cells to various growth factors. Such stem cells are limited in their differentiation potential, producing only neurons and glial cells, including astrocytes and oligodendrocytes (see also Brannen et al., Neuroreport (2000) 11(5): 1123-8; Lillien et al., Dev. (2000) 127: 4993-5005).
The studies described above have shown that totipotent, nearly totipotent, and pluripotent stem cells can be induced to differentiate into specific cell types by manipulating the concentration of growth factors and cytokines in the medium in which they are cultured. Other examples of growth factor-induced differentiation include induction of stem cells to become macrophages, mast cells or neutrophils by IL-3 (Wiles et al., Development (1991) 111:259-267); the direction of cells to the erythroid lineage by IL-6 (Biesecker et al., Exp. Hematol. (1993) 21: 774-778); induction of neuronal differentiation by retinoic acid (Slager et al., Dev. Genet. (1993) 14: 212-224; Bain et al., Dev. Biol. (195) 168:342-357); and induction of myogenesis by transforming growth factor (Rohwedel et al., Dev. Biol. (1994) 164, 87-101). In the latter examples, the inducing agents were not directly applied to ES cells or cells directly derived from the embryo, but rather to aggregates of ES cells or to embryoids.
In addition to manipulating the concentration of growth factors and cytokines, totipotent and pluripotent stem cells may be induced to differentiate into specific cell types by co-culturing them with cells of a different type. For example, Kaufman et al. (U.S. Pat. No. 6,280,718) showed that human ES cells differentiate into hematopoietic precursor cells when cultured on a feeder cell layer of mammalian stromal cells (see col. 5, line 7, to col. 6, line 26). The disclosure of U.S. Pat. No. 6,280,718 is incorporated herein by reference in its entirety. Similarly, Kawasaki et al. have induced the differentiation of cynomolgus monkey ES cells into dopaminergic neurons and pigmented epithelial cells by culturing them on a feeder layer of murine stromal cells (see Proc. Natl. Acad. Sci. USA (2002) 99(3):1580-85).
As shown by the reports described above, research groups' attempts to identify the agents or conditions that induce the differentiation of totipotent and pluripotent stem cells into specific cell types generally involve exposing the stem cells to one or two solutions containing a relatively small number of growth factors or cytokines, and monitoring to see if the stem cells differentiate to acquire a morphology and/or to express a marker gene that is characteristic of a specific cell type.
At present, there is a need for a systematic, large-scale, screening assay to efficiently identify the combinations of biological, biochemical, and physical agents or conditions that act, simultaneously or sequentially, to induce the differentiation of totipotent, nearly totipotent, or pluripotent stem cells into a large number of different, specific cell types.
Also needed are means for efficiently identifying, analyzing and characterizing marker genes and gene products that specifically mark key regulatory steps associated with the induction of differentiation of such stem cells into each of the important specific cell types.
There is also a need for an efficient means for producing and purifying characterized populations of differentiated cells that are suitable and useful for cell therapy, and for testing these in animal models.
The present invention accomplishes these ends, without being limited thereto.
Differentiation Pathways in Oncogenesis
Many molecular events in oncogenesis are a recapitulation or mutation of events that normally occur in differentiation. In this respect, in many cases oncogenesis reflects a reversal of terminal differentiation utilizing, at least in part, pathways used in normal development. Control of cell growth and differentiation by extracellular signals often involves growth factor binding to high affinity transmembrane receptors such as the receptor tyrosine kinases (RTKs) For example, Recently Sakamoto et al, 2001, (Oncol. Rep. 8: 973-80) reported that nerve growth factor and its low-affinity receptor p75NGFR play a role in breast cancer, Gmyrek et al, 2001 (Am. J. Pathol. 159: 579-90) described the role of hepatocyte growth factor/scatter factor ((HGF/SF) that binds the Met receptor and promotes the differentiation of epithelial cells in prostate, kidney, and hepatocellular carcinoma, similarly, mutations in the Ret receptor has been implicated in multiple endocrine neoplasias, the kit receptor in mastocytomas and gastrointestinal tumors, the Flt-3 ligand that plays a role in hematopoietic differentiation has been implicated in neural crest-derived tumors (Timeus et al, 2001, Lab. Invest. 81: 1025-1037), FGF-1 and -2 in pancreatic malignancy (El-Hariry et al, 2001, Br. J. Cancer, 84: 1656-63), HB-EGF in colon cancer (Ito et al, 2001, Anticancer Res. 21: 1391-4), Oncostatin M in breast cancer, Glypicans in breast cancer (Matsuda et al, 2001, Cancer Res. 61: 5562-9), and Yiu et al, 2001 (Am. J. Pathol. 159: 609-22) described the role of the extracellular matrix component SPARC in the apoptosis pathway in ovarian cancer. These only a few examples of the many extracellular components that are important in the differentiation of a particular cell type, and also play a role in cancer. Surprisingly, few assays for antitumor agents, or assays for novel targets in cancer therapy have been based on the identification of factors influencing early differentiation pathways. The present invention also provides means for efficiently screening many combinations of biological, biochemical, and physical agents or conditions to identify treatments that may induce cancerous cells to undergo differentiation and inhibit their proliferation.